Understanding the molecular mechanism by which pluripotency is maintained in human embryonic stem cells (hESC) is important for the development of improved methods to derive, culture and differentiate these into cells of potential therapeutic use. Large-scale transcriptional comparison of the hES-NCL1 line derived from a day 8 embryo with H1 line derived from a day 5 embryo (WiCell Inc.) showed that only 0.52% of the transcripts analysed varied significantly between the two cell lines. This is within the variability range that has been reported when hESC derived from days 5-6 embryos have been compared with each other. This implies that transcriptional differences between the cell lines are likely to reflect their genetic profile rather than the embryonic stage from which they were derived. Bioinformatic analysis of expression changes observed when these cells were induced to differentiate as embryoid bodies suggested that quite a few of the downregulated genes were components of signal transduction networks. Subsequent analysis using western blotting, flow cytometry and antibody arrays implicated components of the PI3K/AKT kinase, MAPK/ERK and NFkappabeta pathways and confirmed that these components are decreased upon differentiation. Disruption of these pathways in isolation using specific inhibitors resulted in loss of pluripotency and/or loss of viability suggesting the importance of such signalling pathways in embryonic stem cell maintenance.

The role of PI3K/AKT, MAPK/ERK and NFκβ signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis

The transcription factor Oct‐4 is expressed specifically in the totipotent germline cycle of mice. Cells that lose Oct‐4 differentiate along different paths to form embryonic and extraembryonic somatic tissue. Oct‐4 may maintain the potency of stem and germline cells by preventing all other differentiation pathways. Oct‐4 may also regulate the molecular differentiation of cells in the germ lineage as it progresses from the fertilized egg, through cleavage stage/morula blastomeres, blastocyst, inner cell mass, epiblast, germ cells, and gametes. The factors that regulate, and are regulated by, Oct‐4 are reviewed with respect to the phenomena of cell potency and germ/soma segregation and differentiation. BioEssays 20:722–732, 1998. © 1998 John Wiley & Sons, Inc.

Errata-Maurizio P, Gross, MK, Scholer HR. 1998. In line with our ancestors: Oct-4 and the mammalian germ

The IL-6 feed-forward loop: a driver of tumorigenesis.

The ability of stem cells to self-renew and to replace mature cells is fundamental to ontogeny and tissue regeneration. Stem cells of the adult organism can be categorized as mono-, bi-, or multipotent, based on the number of mature cell types to which they can give rise. In contrast, pluripotent stem cells of the early embryo have the ability to form every cell type of the adult body. Permanent lines of pluripotent stem cells have been derived from preimplantation embryos (embryonic stem cells), fetal primordial germ cells (embryonic germ cells), and malignant teratocarcinomas (embryonal carcinoma cells). Cultured pluripotent stem cells can easily be manipulated genetically, and they can be matured into adult-type stem cells and terminally differentiated cell types in vitro, thereby, providing powerful model systems for the study of mammalian embryogenesis and disease processes. In addition, human embryonic stem cell lines hold great promise for the development of novel regenerative therapies. To fully utilize the potential of these cells, we must first understand the mechanisms that control pluripotent stem cell fate and function. In recent decades, the microenvironment or niche has emerged as particularly critical for stem cell regulation. In this article, we review how pluripotent stem cell signal transduction mechanisms and transcription factor circuitries integrate information provided by the microenvironment. In addition, we consider the potential existence and location of adult pluripotent stem cell niches, based on the notion that a revealing feature indicating the presence of stem cells in a given tissue is the occurrence of tumors whose characteristics reflect the normal developmental potential of the cognate stem cells.

Pluripotent stem cells and their niches

Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established.

/pdf/derivation-of-porcine-embryonic-stem-like-cells-from-in-4099cahrec.pdf

Derivation of Porcine Embryonic Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos

The standard method for isolation of ES cells from strain 129 mice does not give rise to ES lines of CBA origin. We investigated the effect of inhibition of MEK/ERK signaling in combination with increased stimulation of gp130 signaling on derivation of ES cells from CBA blastocysts. Inhibition of MEKI and MEKII using the drug U0126 and stimulation of gp130 signaling by elevating the level of LIF present gave rise to ES-like lines in 22.6% of explants. No lines arose when MEK was inhibited in the absence of additional LIF stimulation, nor when additional LIF stimulation occurred in the absence of MEK inhibition. Typically for ES cell lines, CBA-derived cells contributed to chimeric mice and differentiated broadly in vitro. Increased levels of gp130 signaling led to similar levels of STAT3 activation in strain 129 and CBA ES cells. We conclude that CBA ES cells have a requirement for additional STAT3 activation.

Increased gp130 signaling in combination with inhibition of the MEK/ERK pathway facilitates embryonic stem cell isolation from normally refractory murine CBA blastocysts.

The isolation of murine embryonic stem (ES) cells has been almost exclusively from the 129 mouse strain. Other mouse strains, such as CBA, have proven refractory to ES cell isolation by conventional means. We previously reported the isolation of 87.5% CBA ES lines by selective ablation of differentiating cells (McWhir et al., 1996). Here, we report the isolation of ES and EG cells from 94% CBA embryos hemizygous and homozygous for a neomycin-resistance transgene under the transcriptional control of the Oct3/4 promoter (Oct/neo). Since expression of the Oct/neo transgene only confers drug resistance to undifferentiated cells of the inner cell mass, selection results in the ablation of differentiating cells from the culture. The efficiency of ES isolation by selective ablation in homozygotes is twice that in heterozygotes. ES isolation frequency in permissive strain 129 embryos is enhanced by treatment with an inhibitor of the extracellular-signal-regulated kinase (ERK) pathway but this effect is not sufficient to permit ES isolation from the CBA strain.

Isolation of murine embryonic stem and embryonic germ cells by selective ablation.

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Increased gp130 signaling in combination with inhibition of the MEK/ERK pathway facilitates embryonic stem cell isolation from normally refractory murine CBA blastocysts.

Isolation of murine embryonic stem and embryonic germ cells by selective ablation.